Although the roles of progesterone and prolactin in mammary gland development have been extensively studied, the roles of Insulin, IGF1 and 1GF2 have received much less attention. The Neville laboratory recently found that a null mutation of the insulin receptor (IR) specifically in the secretory epithelium both impedes proliferative activity in early pregnancy and largely inhibits secretory differentiation in late pregnancy. We hypothesize that this receptor and one or more of its ligands are important modulators of mammary differentiation. Similarly, extensive work with the progesterone receptor (PR) in the mammary gland has shown that progesterone and PR are necessary for alveolar proliferation and development. There is also evidence that IR and PR work together to modulate each other's activity, although almost nothing is known about the mechanisms. In this project Dr. Neville and Dr. Edwards, an expert on PR, will work with two experts in metabolism, Drs. Carrie McCurdy and Paul MacLean, to investigate this new area. In vitro experiments will utilize unique 3D acinar cultures of mammary epithelial cells from virgin and 15 day pregnant mice to elucidate the molecular mechanisms by which these two regulatory pathways direct both proliferation and differentiation in the mammary gland. The investigators will also use mouse models of hypoinsulinemia and hyperinsulinemia to examine how conditions such as starvation, diabetes and obesity impinge on these pathways, and through them on milk secretion. Obesity, an increasingly common condition in most developing countries, is often associated with lactation failure in a population where breastfeeding is likely to be most beneficial to the infant. Understanding the mechanisms by which obesity alters mammary development and lactation competence may provide new therapeutic modalities that will enhance breastfeeding success. RELEVANCE (See instructions): Obesity is a primary health concern worldwide that negatively impacts breastfeeding performance. Normally plasma insulin levels rise during pregnancy; preliminary data from our institution show that the rise is higher in obese women. Our studies offer a possible mechanistic link between the effects of insulin upon mammary development, lactation failure, and breast cancer development providing us with the basis for both diagnostic and potential therapeutic interventions